CN108795956A - Application of the GmMDH12 genes in terms of promoting soybean nodulation nitrogen fixing capacity - Google Patents

Application of the GmMDH12 genes in terms of promoting soybean nodulation nitrogen fixing capacity Download PDF

Info

Publication number
CN108795956A
CN108795956A CN201810421922.8A CN201810421922A CN108795956A CN 108795956 A CN108795956 A CN 108795956A CN 201810421922 A CN201810421922 A CN 201810421922A CN 108795956 A CN108795956 A CN 108795956A
Authority
CN
China
Prior art keywords
gmmdh12
gene
soybean
root
root nodule
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201810421922.8A
Other languages
Chinese (zh)
Other versions
CN108795956B (en
Inventor
田江
陈志坚
廖红
梁翠月
白振龙
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
South China Agricultural University
Original Assignee
South China Agricultural University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by South China Agricultural University filed Critical South China Agricultural University
Priority to CN201810421922.8A priority Critical patent/CN108795956B/en
Publication of CN108795956A publication Critical patent/CN108795956A/en
Application granted granted Critical
Publication of CN108795956B publication Critical patent/CN108795956B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01037—Malate dehydrogenase (1.1.1.37)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Nutrition Science (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention disclosesGmMDH12Application of the gene in terms of promoting soybean nodulation nitrogen fixing capacity.Specifically malate dehydrogenase geneGmMDH12Control the synthesis of root nodule malic acid and its application in terms of promoting legume root system dross.The present invention the study found thatGmMDH12Have the function of the synthesis of regulating and controlling soybean root nodule malic acid, overexpressionGmMDH12The compound endogenous malic acid concentration of plant root nodule of soybean transgene is significantly increased, root nodule number and root nodule biomass are increased, to improve N content of crop tissue and biomass.According to the present invention,GmMDH12Gene has the potentiality that legume biological nitrogen fixation function is improved by transgenic technology, and to improving, crop nutrition content efficiently utilizes and the development of environmentally friendly sustainable agriculture has great theoretical and practical significance.

Description

Application of the GmMDH12 genes in terms of promoting soybean nodulation nitrogen fixing capacity
Technical field
The invention belongs to field of plant genetic.More particularly, toGmMDH12Gene is promoting soybean nodulation Application in terms of nitrogen fixing capacity.
Background technology
Legume root system can form cogeneration system -- the root nodule of mutually beneficial complementation with rhizobium.Host plant can be root The growth of tumor bacterium provides carbon source and suitable growing environment, and rhizobium then carry out biological nitrogen fixation by the effect of azotase, are Host growth provides nitrogen source.Legume inoculation technology is applied to agricultural production, improves crop nitrogen nutrition, is legume section fertilizer The effective measures of volume increase, play an important role (Qin et al., 2012) in agricultural production.Rhizobium leguminosarum Growth, root nodule formation and biological nitrogen fixation be a complicated physiological and biochemical procedure, participated in jointly by many metabolic pathways It completes, including carbon metablism approach (Hern á ndez et al., 2009).Soybean [Glycine max (L.) Merr] A kind of important oil plant and protein crop in worldwide, be edible oil and edible protein important source material and I The most important legume of state.Soybean can form root nodule with rhizobium mutualism, can improve the nitrogen nutrition of soybean, reduce Nitrogenous fertilizer is put into.
During root nodule carbon metablism, phosphoenolpyruvate (Phosphoenolpyruvate, PEP) is a weight The mesostate wanted.In root nodule, PEP is mainly assimilated by two approach, and one is that synthesis organic acid enters root nodule Krebs cycle pathway, another is then the amino acid metabolism approach (White et al., 2007) into nitrogen assimilation.It grinds Study carefully and think, in rhizobium, the Main way of carbon metablism is to generate oxaloacetic acid (Oxaloacetate, OAA) through PEP, most The approach (Le Roux et al., 2008) of synthesizing apple acid afterwards.Pass through14C flag is tested, and finds the malic acid in root nodule With marking signal, imply that malic acid may be accumulated as the product of carbon metablism in root nodule, malic acid is considered as rhizobium Important carbon source during respiratory metabolism, be further used for rhizobium metabolic exhaustion (Rosendahl et al., 1990; Le Roux et al., 2006)。
Malate dehydrogenase gene MDH (EC.1.1.1.37) can be catalyzed malic acid and the reversible transition of OAA, be tricarboxylic Key enzyme in sour cycle approach.In the legumes such as clover, pea and Kidney bean, it is found that Rhizobium Inoculation can significantly improve MDH enzymatic activitys, and the increase of MDH enzymatic activitys is considered mainly acting on malic acid in regulation and control root nodule carbon metablism approach in root nodule Synthesis (Miller et al., 1998; Colebatch et al., 2002; Le Roux et al., 2008; Hern ández et al., 2009).Also, numerous studies also indicate that, overexpressionMDHGene can increase clover, soybean and cigarette The endogenous malic acid of the plant such as grass content (Tesfaye et al., 2001; Lü et al., 2012).But regulation and control root Whether the important gene of tumor malic acid synthesis, and the synthesis of increase malic acid can promote root system dross, improve legume Nitrogen nutrition it is still unclear.
Invention content
The technical problem to be solved by the present invention is to overcome the defect of the above-mentioned prior art and deficiency, by proteomics and The analysis methods such as quantitative PCR, the homologous clone one in soybean nodulationGmMDH12Gene（Sequence such as SEQ ID NO.3 institutes Show, encoding amino acid sequence is as shown in SEQ ID NO.4）, find it on albumen and transcriptional level simultaneously by low-phosphorous enhancing Expression, it was demonstrated thatGmMDH12The albumen of gene code has the biological function for promoting the synthesis of root nodule malic acid, and then promotes Root system dross, and finally improve transgenic line nodule nitrogen fixation ability and biomass.
The object of the present invention is to provideGmMDH12Application of the gene in terms of promoting soybean nodulation nitrogen fixing capacity.
Another object of the present invention is to provideGmMDH12Application in terms of genes amplification root nodule malic acid synthesis.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
The present invention relates to the function and applications of a gene.Specifically, present invention offer is a kind of to utilize malate dehydrogenase geneGmMDH12Increase the synthesis of Legume nodule malic acid, the method for promoting nodulation and nitrogen fixation, improving plant nitrogen efficiency passes through overexpressionGmMDH12, the compound plant of genetically engineered soybean, which has, increases the endogenous malic acid concentration of root nodule, increases root nodule numbers and biomass, with And improve the phenotype of N content of crop tissue.
The present invention has carried out malic dehydrogenase firstGmMDH12The clone of full length gene cDNA, and grind as follows Study carefully
（1）Using e. coli protein expression and purification system, GmMDH12 zymologic properties are analyzed；Using structure soybean transgene Compound plant method, analyzes in soybean hair rootGmMDH12The tissue of gene positions, the overexpression in soybean hair rootGmMDH12, comparative studiesGmMDH12Transgenic line with compare phenotype of the strain in root system dross etc..
（2）It obtainsGmMDH12Promoter drivesGUSThe compound plant of soybean transgene of reporter gene, analyzesGmMDH12The Histochemical localization of gene.
（3）Obtain GmMDH12-GST fusion proteins.
（4）Obtain overexpressionGmMDH12The compound plant of soybean transgene.
Result of study of the present invention is shown：
（1）The Histochemical localization of gene of the present invention analysis shows,GmMDH12Be primarily targeted for bacteroid infected cell, In the root nodules tissue such as vascular bundle and cortex.
（2）It is of the present inventionGmMDH12The albumen of gene code has the work of catalysed in vitro oxaloacetic acid synthesizing apple acid Property.
（3）The compound plant root nodule malic acid concentration of overexpression transgenosis of gene of the present invention is significantly higher than control strain System, i.e. overexpressionGmMDH12Increase the compound endogenous malic acid concentration of plant root nodule of transgenosis.
（4）Under the conditions of the processing of low nitrogen and Rhizobium Inoculation, the compound plant of overexpression transgenosis of gene of the present invention The root nodule numbers and biomass of strain are all remarkably higher than control strain, and N content of crop tissue and dry weight are significantly higher than control strain.I.e. OverexpressionGmMDH12Increase the compound plant root nodule number of transgenosis and root nodule biomass, increase the compound plant weights of transgenosis and Nitrogen content.
（5）It is right that the present invention passes throughGmMDH12In the functional analysis of the compound plant of soybean transgene, specifyGmMDH12? Function in Legume nodule growth course, illustratesGmMDH12The root system dross mechanism of participation is that the pulse family including soybean is made Object nitrogen has the application prospect of the fertile high yield of section, to development environment friendly efficiently using genetic resources and theoretical foundation are provided Sustainable agriculture has important practice significance.
Therefore, applying below should all be within protection scope of the present invention：
GmMDH12Application of the gene in terms of enhancing legume nodule malic acid synthesis.
GmMDH12Application of the gene in terms of promoting legume nodulation nitrogen fixing capacity.
GmMDH12Application of the gene in terms of the strong legume of cultivation root system Noduling ability and/or biological nitrogen fixation ability.
Preferably, the legume is soybean.
In addition, based on the present invention, additionally provide a kind of structure root system Noduling ability and/or biological nitrogen fixation ability it is strong turn base Because of the method for soybean, be byGmMDH12Gene is overexpressed in soybean.
Specifically, overexpression is builtGmMDH12The method of transgenosis compound soybean is as follows：
S1. overexpression carrier is built
Using soybean nodulation cDNA as template, with SEQ ID NO:14 and SEQ ID NO:Upstream and downstream specific primer PCR shown in 15 expands IncreaseGmMDH12Gene order；
After pcr amplified fragment recycling sequencing is errorless, pass throughSac IAnd XbaI is to amplified fragments and purpose carrier PTF101S difference It, will after carrying out double digestionGmMDH12Gene amplification fragment is connected on purpose carrier PTF101S, obtains expression vector；
S2. it converts
Then the expression vector built is transferred in K599 by freeze-thaw method, it is compound using agriculture bacillus mediated soybean transgene Plant is converted, and the compound plant of transgenosis is obtained；
S3. it verifies
The compound plant root of extraction transgenosis and root nodule RNA, reverse transcription pass through quantitative PCR detection at cDNAGmMDH12Gene table Up to effect.
The invention has the advantages that：
The present invention is the study found that malate dehydrogenase geneGmMDH12Function in terms of soybean nodulation growth metabolism：GmMDH12There is expression in the root nodules tissues such as the infected cell, vascular bundle and cortex of mature nodule bacteroid, and with root nodule The extension of growth time, the gene expression amount gradually increase.Function in terms of soybean nodulation malic acid synthesis：GmMDH12 exists It is external to have the function of being catalyzed oxaloacetic acid synthesizing apple acid.Also, compared with the control, overexpressionGmMDH12It dramatically increases Soybean transgene compound plant root nodule malic acid concentration.Function in terms of promoting root system dross：Compared with the control, excess table It reachesGmMDH12The root nodule number and root nodule biomass for significantly increasing the compound plant of soybean transgene, improve N content of crop tissue and Biomass.
It thus provides geneGmMDH12Increase the synthesis of soybean nodulation malic acid, promote soybean nodulation fixed nitrogen, cultivation New opplication in terms of efficient nodulation and nitrogen fixation transgenic leguminous plants and raising crop nitrogen efficiency can be to improve including soybean Legume biological nitrogen fixation ability and nitrogen efficiently provide candidate gene resource using the molecular breeding with high yield.According to this hair It is bright,GmMDH12Gene has the potentiality that legume biological nitrogen fixation function is improved by transgenic technology, to improving crop nutrition content It efficiently utilizes and the development of environmentally friendly sustainable agriculture has great theoretical and practical significance.
Description of the drawings
Fig. 1：GmMDH12Albumen and gene expression analysis.
Fig. 2：GmMDH12Promoter drivesGUSExpressive site analysis.
Fig. 3：GmMDH12 characterization analysis.
Fig. 4：OverexpressionGmMDH12Influence to the compound plant dross of soybean transgene.
Fig. 5：OverexpressionGmMDH12Influence to soybean transgene compound plant strain growth and nitrogen/phosphorus content.
Specific implementation mode
It is further illustrated the present invention below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art Agent, method and apparatus.
Unless stated otherwise, following embodiment agents useful for same and material are purchased in market.
Embodiment 1GmMDH12Gene diffusion is analyzed
1, GmMDH12 protein expression analysis
Root nodule proteomic assays experimental design：After soybean varieties " HN66 " seed is sprouted 5 days, seedling inoculation rhizobium BXYD3 1 hour, it is then transferred to low-phosphorous (LP, 5 μM of KH2PO4) or high phosphorus (HP, 250 μM of KH2PO4) Poor nitrogen nutrition liquid (50 μM NH4NO3) in, after cultivating 25 days, harvest overground part, root and root nodule sample, and in -80 DEG C of preservations, it is used for root nodule The extraction of albumen and RNA.Different time points gene expression analysis experimental design：After soybean HN66 seeds are sprouted 5 days, seedling inoculation Rhizobium BXYD3 1 hour is then transferred to low-phosphorous (LP, 25 μM of KH2PO4) or high phosphorus (HP, 500 μM of KH2PO4) Poor nitrogen nutrition liquid (250 μM of NH4NO3) in culture harvested root and root respectively in the 14th, 21,30 and 40 day of culture Tumor sample is used for the extraction of malic acid and RNA in -80 DEG C of preservations.
Two-dimensional Electrophoresis Analysis：3 g root nodule samples addition, 8.0 mL Extraction buffers (50 % phenol, 0.45 M sucrose, 25 MM EDTA, 2 % beta -mercaptoethanols, 250 mM Tris-HCl pH 8.8,2 mM PMSF, 2 % PVPP, 0.3-1.4 M NaCl, 10-40 % ethyl alcohol) grinding, mixing, 12000 rpm are centrifuged 15 minutes at 4 DEG C, draw upper layer phenol solution, are added 1/10 times of 5 M NaCl of volume is added the methanol (containing 0.1 M ammonium acetates and 1 % beta -mercaptoethanols) of 5 times of volumes precooling, mixes Even, after -20 DEG C stand 2 hours, 12000 rpm are centrifuged 15 minutes at 4 DEG C, abandon supernatant, and the methanol of precipitation precooling (contains 0.1 M ammonium acetates) it rinses 2 times, it is then washed 2 times with 80 % acetone, is finally washed 1 time with 70 % alcohol, collect precipitation, vacuum is dry Dry, -80 DEG C save backup.Protein example be added 600 μ L lysates (7 M urea, 2 M thiocarbamides, 4 % CHAPS, 80 MMDTT, 1 % IPG Buffer) it is sufficiently mixed, vortex oscillation 15 minutes, 12000 rpm are centrifuged 15 minutes at 4 DEG C, on It is protein example liquid clearly.It is right using protein quantification kit (2-D Quant-Kit, GE-Healthcare, USA) Protein example carries out concentration mensuration, prepares bovine serum albumin BSA (GE-Healthcare, USA) standard curve, measures sample Product absorbance value calculates sample protein matter concentration.
430 μ L protein sample liquid are added into adhesive tape groove, is put into IPG adhesive tape (24 centimetres) and covering oil is added dropwise afterwards, cover Adhesive tape groove lid is placed on IPGphor (GE-Healthcare, USA), and isoelectric focusing (IEF) program is 25 DEG C, 50 μ A glue-1, voltage and time parameter are：(linear 2 is small by 30 V (quick 12 hours) → 50 V (quick 2 hours) → 100 V When) → 200 V (linear 2 hours) → 500 V (linear 2 hours) → 1000 V (linear 2 hours) → 8000 V (linear 4 Hour) → 8000 V (quick 90000 volts of hours) → 500 V (quick 10 hours).The IPG adhesive tape completed will be focused to put down Weighing apparatus.Adhesive tape balance is carried out in two steps：Equilibrium liquid [equilibrium liquid of the first step containing 1 % DTT: 50 mM Tris-HCl (pH 8.8), 6 M urea, 30 % (V/V) glycerine, 2 % (W/V) SDS, micro bromophenol blue] it balances 15 minutes；Second step is with containing There is the equilibrium liquid of 2.5 % iodoacetamides to balance 15 minutes.
The IPG adhesive tape balanced moves to gel upper end, be put into Ettan DALTsix electrophoresis apparatuses (GE-Healthcare, USA electrophoresis, 10 mA glue) are carried out-1After electrophoresis 1 hour, 40 mA glue-1Electrophoresis is to terminating.After electrophoresis, deionization is used Water rinses gel, after fixer [40 % (V/V) absolute ethyl alcohol, 10 % (V/V) glacial acetic acid] fixes 1 hour or more, uses Coomassie brilliant G-250 dyeing liquor [0.08 % (W/V) Coomassie brilliant G-250,25 % (V/V) methanol, 1 % (V/V) Phosphoric acid] stained over night, is then rinsed for several times with deionized water, until gel clear background, is swept with 1640 ' L-types of EPSON Instrument is retouched to be scanned.2-DE glue is analyzed using (Bio-Rad, the USA) softwares of PD-Quest 7.0, is filtered out low Phosphorus and the root nodule protein site of the lower significant difference expression of high phosphorus processing use after trypsase (Trysin) film dosim 4700 TOF-TOF mass spectrographs of ABI (Applied Biosystems, USA) carry out MALDI-TOF/TOF MS mass spectrums point Analysis.
2、GmMDH12Gene diffusion is analyzed
Homologous comparison is carried out on soybean genome database Phytozome, it is shared to predict soybean malate dehydrogenase enzyme family 16 members, binding protein group credit analysis, determine to be regulated and controled by phosphorus in root noduleGmMDH12Gene and protein sequence information, designGmMDH12Full length gene primer, upstream specific primer 5 '-ATGATGAAGCCATCGATGCTCAGAT-3 ' (SEQ ID NO: 1), 5 '-TTACTGGTTGGCAAATTTGATTCCCTT-3 ' of downstream special primer (SEQ ID NO:2), with soybean nodulation cDNA For template, PCR amplificationGmMDH12Gene open reading frame (ORF) full length sequence, and comparison is sequenced, it obtainsGmMDH12Gene ORF full length sequences are 1038 bp (SEQ ID NO:3) 345 amino acid residue (SEQ ID NO, are encoded:4), protein molecular Amount is 36 kD.
The soybean nodulation RNA for extracting different time points harvest makees after the cDNA obtained by RNA sample reverse transcription is diluted 50 times For the template of quantitative PCR reaction.Quantitative PCR analysis is carried out with SYBR Green (Takara, Japan) quantification kit, It is reacted with 3000 qRT-PCR systems of Rotor-Gene (Corbett Research, Australia).20 μ L are anti- It is that 10 μ L 2 × SYBR Green PCR master mix, 6.4 μ L Mili-Q water, 0.8 10 μM of μ L's draws to answer system Object, 2 μ L dilute 100 times of cDNA templates.Response procedures are, 95 DEG C 1 minute, 94 DEG C 15 seconds, 60 DEG C 15 seconds, 72 DEG C 30 seconds, 40 cycles.With soybeanGmEF1aAs house-keeping gene, relative expression quantity=destination gene expression amount/house-keeping gene (GmEF1a) expression quantity.GmMDH12The primer of gene quantification PCR is：F: 5'-CTCACTCACTAATCGCCACTCTCAC-3' (SEQ ID NO:5), R: 5'-GCGGAGTGGAGAGATCTGAGCA-3' (SEQ ID NO:6).Soybean house-keeping geneGmEF1aQuantification PCR primer is：F: 5'-TGCAAAGGAGGCTGCTAACT-3' (SEQ ID NO:7), R: 5'- CAGCATCACCGTTCTTCAAA-3’ (SEQ ID NO:8)。
3, the endogenous malic acid extraction of soybean nodulation and measurement
0.2 gram of soybean nodulation sample is taken, is ground with 0.6 mL, 0.5 M HCl, then distilled and washed after mortar together with 0.6 mL Be transferred to centrifuge tube, during which 80 DEG C of water-baths 20 minutes mix well several times, 4 DEG C, 12000 rpm are centrifuged 15 minutes, suct it is clear, 1 mL supernatants are taken to cross 0.45 μm of miillpore filter.Using 1260 Infinity LC series of high performance liquid chromatograph (Agilent, USA), 35 DEG C of column temperature, mobile phase are 0.2 % metaphosphoric acids, and sample passes through C18 columns (mm of 4.6 mm × 250) Afterwards, the absorption value of 220 nm of Detection wavelength calculates the concentration of malic acid in sample by making malic acid standard curve.
4, experimental result
Fig. 1 isGmMDH12Albumen and gene expression analysis.Wherein A：Expression of the GmMDH12 albumen in root nodule；B：GmMDH12 Expression of the gene in root and root nodule；C：GmMDH12Expression in different development stage root nodule；D：Different development stage root nodule Endogenous malic acid concentration.Data are the average value and standard error that 3 biology repeat in figure.The result shows that：GmMDH12Albumen With gene by phosphorus nutrient regulating and expressing (Fig. 1 A, B) in root nodule.As shown in Fig. 1 C, under the processing of high and low phosphorus, with The extension of Nodule Growth time,GmMDH12Gene expression amount gradually increases, the expression quantity highest at 30 days, and at 40 days Root nodule in expression quantity decrease.WithGmMDH12Gene expression pattern is similar, and with the extension of growth time, root nodule is endogenous Malic acid concentration presentation first increases the trend reduced afterwards, the malic acid concentration highest in 21 days root nodules, in 40 days aging roots It is minimum in tumor.The result shows thatGmMDH12Take part in the growth metabolism process of root nodule.
Embodiment 2 GmMDH12Gene promoter clone, vector construction and tissue positioning analysis
1, experimental method
GmMDH12Gene promoter is cloned and GUS expression vector establishments：Soybean root genomic DNA is extracted, with soybean genome DNA is template, with upstream specific primer 5 '-GGGATCCCCGTGCATGTGTTGA-3 ' (SEQ ID NO:9) and downstream is special Primer 5 '-ATGAATTCAGTGGCGATTAGTGAG-3 ' (SEQ ID NO:10) it expandsGmMDH122182 bp of promoter (SEQ ID NO:11), after PCR fragment recycling sequencing, pass throughBamH IAnd EcoRI is to recycling segment and purpose carrier After pCAMBIA1391 carries out double digestion, promoter fragment is connected on pCAMBIA1391, will then be built by freeze-thaw method Good GUS expression vectors are transferred in Agrobacterium rhyzogenesK599 that (K599 bacterial strains are ground by University of Queensland's pulse family synthesis Study carefully center to give, be stored in root system biological study central laboratory of Agricultural University Of South China, specific descriptions are shown in document Kereszt et al., 2007)。
The structure of the compound plant of agriculture bacillus mediated soybean transgene：Soybean varieties HN66 seeds are sprouted 5 days in sand culture It is used for the induction of hair root afterwards, is stained with above-mentioned cultured K599 thalline with 1 mL syringes, is pricked back and forth in seedling hypocotyl position Thorn, and it is coated with thalline in wound, so that thalline is fully infected wound.Seedling after infecting is transferred in normal nutrition liquid and is trained It supports, visible hair root is grown from treatment of wounds after about 14 days, during which pays attention to the humidity for keeping infecting wound.It is gone after hair root is grown Except main root, hair root is placed in nutrient solution and continues to cultivate.It is small to hair root Rhizobium Inoculation 1 when hair root grows 10 centimetres or so When, in low nitrogen (50 mM NH4NO3) and low-phosphorous (5 μM of KH2PO4) cultivate 21 and 30 days in nutrient solution after, carry out GUS dyes Color and paraffin section analyze tissue positioning scenarios of the gene in root and root nodule.
2, experimental result
Fig. 2 isGmMDH12Promoter drivesGUSExpressive site analysis.The result shows that in root,GUSGene is mainly in root Point and center pillar etc. express (Fig. 2 B-D, scale are 10 mm) in positions.Fig. 2 E and 2F respectively be growth 21 days root nodules it is longitudinal sectional and Sectional view, Fig. 2 G are 30 days root nodule sectional views of growth, and Fig. 2 H are Fig. 2 G partial enlarged views, and scale is 200 mm.
The result shows that in root nodule,GUSGene is mainly expressed in root nodule bacteroid, vascular bundle and cortex detail, and with The extension for the Nodule Growth time, the expression quantity in bacteroid infected cell gradually increase, showGmMDH12Take part in root nodule Growth course.
3 GmMDH12 characterization analysis of embodiment
1, experimental method
（1）The structure of prokaryotic expression carrier：Using soybean nodulation cDNA as template, with upstream specific primer 5 '- GGATCCATGATGAAGCCATCGA-3' (SEQ ID NO:And downstream special primer 5 ' -12) ACATGAATTCTTACTGGTTGGCAAATT-3' (SEQ ID NO:13) it expandsGmMDH12Full length gene, PCR fragment recycling Be sequenced it is errorless after, pass throughBamH IAnd EcoRI to segment and prokaryotic expression carrier pGEX6P-3 (GE-Healthcare, It USA, will after) carrying out double digestionGmMDH12Gene is connected on purpose vector pGEX 6P-3, will then be built by freeze-thaw method Good expression vector is transferred in BL21 Escherichia coli.
（2）It takes in the appropriate above-mentioned BL21 bacterium solutions to LB liquid medium fresh 200 mL containing target gene in 28 DEG C culture to OD600 be 0.4, be then added 0.2 mM IPTG continue culture 12 hours, bacterium solution in 12000 rpm centrifuge 10 points Clock collects thalline, with 8 mL Binding buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) and precipitation is resuspended, ultrasonic wave size degradation thalline is used in combination, centrifuges, collects supernatant, supernatant and 2 mL Glutathione Sepharose 4B (GE-Healthcare, USA) react 3 hours at 4 DEG C, then use Binding Buffer elutes foreign protein 3 times, finally uses Elution buffer (50 mM Tris-HCl, 10 mM Reduced Glutathione, pH 8.0) elute column 3 times, collect GmMDH12 destination proteins.
（3）To the measurement of optimal reactive temperature, reaction mixture is firstly placed in different temperatures (20 DEG C -90 DEG C) temperature It educates after ten minutes, in the enzyme for adding equivalent when reaction carries out, mixing is placed in ultraviolet specrophotometer and measures in 2 minutes The changing value of OD340 records data situation.To the measurement of optimal reaction pH, 100 mM Tris-HCl in mixture are matched It is 7.0-9.0 difference gradients that pH, which is made at identical temperature, then prepares reaction mixture, and when activity analysis is added enzyme solution, after mixing It is placed in ultraviolet specrophotometer and measures the changing value of OD340 in 2 minutes, record data situation.To different concentration of substrate Measurement, different OAA concentration of substrate (0.02-3 mM) is set, is mixed in the enzyme and reaction for adding equivalent when reaction carries out Object, mixing are placed on the changing value that OD340 in 2 minutes is measured in ultraviolet specrophotometer, record data situation, obtain Data acquire the K of zymetology reaction using double methods (Lineweaver-Burk) reciprocalmValue.Different metal ions are to enzymatic activity Impact analysis, respectively at the different metal ions (Mn that 0.1 mM is added in the enzyme and reaction mixture of equivalent2+、Zn2+、Mg2+、 Fe2+、Cu2+、Al3+、Ag2+And Hg2+), mixing is placed on the changing value that OD340 in 2 minutes is measured in ultraviolet specrophotometer.With Catalysis reaction 1 μm of ol of NADH per minute is defined as 1 unit of enzyme activity (U), 6.22 L of molar extinction coefficient of NADH mmol·cm-1。
2, experimental result
Fig. 3 is GmMDH12 albumen characterization analysis.Wherein, A：Different OAA concentration of substrate influence enzymatic activity；B：Different pH Value influences enzymatic activity；C：Different temperatures influences enzymatic activity；D：Different metal ions influence enzymatic activity.
As a result it shows：The most suitable concentration of substrate of GmMDH12 albumen is 1.5 mM OAA, the K of enzymatic reactionmValue is 0.434 mM (Fig. 3 A), optimal reaction pH value and temperature are respectively pH 8.0 and 50 DEG C (Fig. 3 B, C), also, 0.1 mM Mg2+、Ag+And Hg+Ion significantly suppresses the enzymatic activity (Fig. 3 D) of GmMDH12.
4 overexpression of embodimentGmMDH12Vector construction, the acquisition and detection of the compound plant of soybean transgene
The structure of overexpression carrier：Using soybean nodulation cDNA as template, with upstream specific primer 5 '- ACAAAGAGCTCGAGAGAGAGAAA-3' (SEQ ID NO:And downstream special primer 5 ' -14) ATATCTAGAAACCTACCAGACATGTATG-3' (SEQ ID NO:15) it expandsGmMDH12Sequence, PCR fragment recycling sequencing After errorless, pass throughSac IAnd XbaIt, will after I carries out double digestion to segment and purpose carrier PTF101SGmMDH12Gene connects Onto purpose carrier PTF101S, then the expression vector built is transferred in K599 by freeze-thaw method, by above-mentioned agriculture bar Bacterium mediates the compound plant of soybean transgene to be converted, and is control (CK) to turn empty carrier PTF101S strains.
The compound plant root of extraction transgenosis and root nodule RNA, reverse transcription further pass through quantitative PCR detection at cDNAGmMDH12In the effect of gene expression, detection method and process and above-described embodiment oneGmMDH12Gene Expression Profile Analysis It is identical.
5 overexpression of embodimentGmMDH12Influence to the compound plant dross of soybean transgene
1, experimental method
When the 4 compound plant of gained soybean transgene of example to be performed and control strain hair root grow 10 cm, Rhizobium Inoculation is carried out respectively BXYD3 1 hour is then transferred to low-phosphorous (LP, 5 μM of KH2PO4) or high phosphorus (HP, 250 μM of KH2PO4) low nitrogen Nutrient solution (50 μM of NH4NO3) in, after cultivating 30 days, harvest overground part, root and root nodule sample, and measure relevant physiological and refer to Mark, including：Root nodule number and root nodule fresh weight, root nodule malic acid concentration.
2, experimental result
Fig. 4 is overexpressionGmMDH12Influence to the compound plant dross of soybean transgene.Wherein, A：Root nodule phenotype, scale are 1 cm；B：Transgenosis root noduleGmMDH12Expression analysis；C：The endogenous malic acid concentration of transgenosis root nodule；D：Root nodule fresh weight；E：Root nodule Number.CK indicates empty vector control strain；OX indicates overexpressionGmMDH12The compound plant of transgenosis.Test data is 5 biologies Learn the average value and standard error repeated.* number expression compares the significant difference between CK strains and OX strains,p< 0.05；ns：Table Show that difference is not notable.
As a result it shows：Opposite empty vector control strain CK, overexpressionGmMDH12Significantly increase the compound plant of transgenosis The endogenous malic acid concentration of root nodule (Fig. 4 C) and root nodule number and root nodule biomass (Fig. 4 D, E).
6 excess of embodimentGmMDH12Influence to soybean transgene compound plant strain growth and nitrogen/phosphorus content
1, experimental method
When the compound plant of soybean transgene and control strain hair root grow 10 cm, Rhizobium Inoculation 1 hour, is then shifted respectively To low-phosphorous (LP, 5 μM of KH2PO4) or high phosphorus (HP, 250 μM of KH2PO4) Poor nitrogen nutrition liquid (50 μM of NH4NO3) In, after cultivating 30 days, overground part and root are harvested, and measure relative physiologic index, including：Biomass and nitrogen and phosphorus content.
Biomass measures：The overground part and root samples of harvest finish 30 minutes in 105 DEG C of baking ovens, then in 75 DEG C drying to constant weight, weighs dry weight.
Plant nitrogen, phosphorus measure：0.1 g of overground part and root samples or so is weighed respectively, the 5 mL concentrated sulfuric acids of addition, which disappear, to be boiled, Then distilled water constant volume is used to cause 50 mL, after sample is diluted 4 times, being placed in Continuous Flow Analysis instrument, (model SAN++, is originated from Holland) in be measured.Plant nitrogen and phosphorus content is calculated according to the concentration of nitrogen and phosphorus of measured sample and plant weights.
2, experimental result
Fig. 5 is overexpressionGmMDH12Influence to soybean transgene compound plant strain growth and nitrogen/phosphorus content.Wherein, A：Plant Dry weight；B：N content of crop tissue；C：Plant phosphorus content.The compound plant Rhizobium Inoculation of soybean transgene is transferred to low-phosphorous after 1 hour (LP, 5μM KH2PO4) or high phosphorus (HP, 250 μM of KH2PO4) Poor nitrogen nutrition liquid (50 μM of NH4NO3) in culture 30 days.CK indicates empty vector control strain；OX indicates overexpressionGmMDH12The compound plant of transgenosis.Test data is 5 lifes The average value and standard error that object repeats.* number expression compares the significant difference between CK strains and OX strains,p< 0.05；ns： Indicate that difference is not notable.
The result shows that：Opposite empty vector control strain CK, overexpressionGmMDH12Significantly increase the compound plant of transgenosis Dry weight (Fig. 5 A) and nitrogen content (Fig. 5 B), but phosphorus content is had no significant effect (Fig. 5 C).
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications, Equivalent substitute mode is should be, is included within the scope of the present invention.
Sequence table
<110>Agricultural University Of South China
<120>Application of the GmMDH12 genes in terms of promoting soybean nodulation nitrogen fixing capacity
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213>Expand GmMDH12 full length genes upstream specific primer (forward primer)
<400> 1
atgatgaagc catcgatgct cagat 25
<210> 2
<211> 27
<212> DNA
<213>Expand GmMDH12 full length genes downstream special primer (reverse primer)
<400> 2
ttactggttg gcaaatttga ttccctt 27
<210> 3
<211> 1038
<212> DNA
<213>Gene GmMDH12 sequences (GmMDH12)
<400> 3
atgatgaagc catcgatgct cagatctctc cactccgccg ccacccgcgg cgcctctcat 60
ctcttccgcc gtgggtacgc ctccgagccg gtgccggagc gcaaggtagc cgttctcggc 120
gccgccggcg ggatcgggca gcctctctcc cttctcatga agctcaatcc cctcgtttcg 180
agcctctccc tttacgatat cgccggaact cccggcgtcg ccgccgatat cagccacata 240
aacaccagat ctgaggtagt ggggtaccaa ggtgatgaag agcttggaaa agctttggag 300
ggtgccgatg ttgttataat tcctgctggt gtgcccagaa agcctggaat gactcgtgat 360
gatcttttta acatcaatgc tggcattgtt aagacgctgt gtactgctat tgctaagtat 420
tgcccccatg cccttgttaa catgataagc aatcctgtga actccactgt tcctattgct 480
gctgaagttt tcaagaaggc aggaacttat gatgagaaga gattgtttgg tgttaccacc 540
cttgatgttg ttagggcaaa aactttctat gctgggaaag ccaatgttcc agttgctggt 600
gttaatgtac ctgttgtggg cggccatgca ggcattacta ttctgccact attttctcaa 660
gccacaccaa aagccaatct tgatgatgat gtcattaagg ctcttacaaa gaggacacaa 720
gatggaggaa cagaagttgt agaagctaag gctggaaagg gttctgcaac tttgtcaatg 780
gcctatgctg gtgccctttt tgctgatgct tgcctcaagg gcctcaatgg agtcccagat 840
gttgtcgagt gctctttcgt gcaatccact gttactgaac ttcccttctt tgcttccaag 900
gtgaggcttg ggacggttgg tgtggaggaa gttctgggct tggggcacct ctcagatttt 960
gagcaacaag gcctcgaaag ccttaagcct gaactcaaat catcaattga gaagggaatc 1020
aaatttgcca accagtaa 1038
<210> 4
<211> 345
<212> PRT
<213>Gene GmMDH12 coding protein sequences (GmMDH12)
<400> 4
Met Met Lys Pro Ser Met Leu Arg Ser Leu His Ser Ala Ala Thr Arg
1 5 10 15
Gly Ala Ser His Leu Phe Arg Arg Gly Tyr Ala Ser Glu Pro Val Pro
20 25 30
Glu Arg Lys Val Ala Val Leu Gly Ala Ala Gly Gly Ile Gly Gln Pro
35 40 45
Leu Ser Leu Leu Met Lys Leu Asn Pro Leu Val Ser Ser Leu Ser Leu
50 55 60
Tyr Asp Ile Ala Gly Thr Pro Gly Val Ala Ala Asp Ile Ser His Ile
65 70 75 80
Asn Thr Arg Ser Glu Val Val Gly Tyr Gln Gly Asp Glu Glu Leu Gly
85 90 95
Lys Ala Leu Glu Gly Ala Asp Val Val Ile Ile Pro Ala Gly Val Pro
100 105 110
Arg Lys Pro Gly Met Thr Arg Asp Asp Leu Phe Asn Ile Asn Ala Gly
115 120 125
Ile Val Lys Thr Leu Cys Thr Ala Ile Ala Lys Tyr Cys Pro His Ala
130 135 140
Leu Val Asn Met Ile Ser Asn Pro Val Asn Ser Thr Val Pro Ile Ala
145 150 155 160
Ala Glu Val Phe Lys Lys Ala Gly Thr Tyr Asp Glu Lys Arg Leu Phe
165 170 175
Gly Val Thr Thr Leu Asp Val Val Arg Ala Lys Thr Phe Tyr Ala Gly
180 185 190
Lys Ala Asn Val Pro Val Ala Gly Val Asn Val Pro Val Val Gly Gly
195 200 205
His Ala Gly Ile Thr Ile Leu Pro Leu Phe Ser Gln Ala Thr Pro Lys
210 215 220
Ala Asn Leu Asp Asp Asp Val Ile Lys Ala Leu Thr Lys Arg Thr Gln
225 230 235 240
Asp Gly Gly Thr Glu Val Val Glu Ala Lys Ala Gly Lys Gly Ser Ala
245 250 255
Thr Leu Ser Met Ala Tyr Ala Gly Ala Leu Phe Ala Asp Ala Cys Leu
260 265 270
Lys Gly Leu Asn Gly Val Pro Asp Val Val Glu Cys Ser Phe Val Gln
275 280 285
Ser Thr Val Thr Glu Leu Pro Phe Phe Ala Ser Lys Val Arg Leu Gly
290 295 300
Thr Val Gly Val Glu Glu Val Leu Gly Leu Gly His Leu Ser Asp Phe
305 310 315 320
Glu Gln Gln Gly Leu Glu Ser Leu Lys Pro Glu Leu Lys Ser Ser Ile
325 330 335
Glu Lys Gly Ile Lys Phe Ala Asn Gln
340 345
<210> 5
<211> 25
<212> DNA
<213>The primers F (forward primer F) of GmMDH12 gene quantifications PCR
<400> 5
ctcactcact aatcgccact ctcac 25
<210> 6
<211> 22
<212> DNA
<213>The primer R (reverse primer R) of GmMDH12 gene quantifications PCR
<400> 6
gcggagtgga gagatctgag ca 22
<210> 7
<211> 20
<212> DNA
<213>Soybean house-keeping gene GmEF1a quantification PCR primers F (forward primer F)
<400> 7
tgcaaaggag gctgctaact 20
<210> 8
<211> 20
<212> DNA
<213>Soybean house-keeping gene GmEF1a quantification PCR primers R (reverse primer R)
<400> 8
cagcatcacc gttcttcaaa 20
<210> 9
<211> 22
<212> DNA
<213>Expand GmMDH12 promoters upstream specific primer (forward primer F)
<400> 9
gggatccccg tgcatgtgtt ga 22
<210> 10
<211> 24
<212> DNA
<213>Expand GmMDH12 promoters downstream special primer (reverse primer R)
<400> 10
atgaattcag tggcgattag tgag 24
<210> 11
<211> 2182
<212> DNA
<213>GmMDH12 promoter sequences (GmMDH12 Promoter)
<400> 11
cccgtgcatg tgttgatagt agttatgaat ttttaagatt aaaaactaaa acttcattta 60
ggcatttcat caaacacata acttacaacc catatcggga agatgacgaa aaaattagcc 120
cgggaaaata aaaaaggaac caaaaacaga ggctaaaaat cagtaagaaa aaataattat 180
tattaagaag aaaacatgca taacaacaaa caaaattaga accttgcaaa taaaaataaa 240
tagtaatgaa acaaacatgc ataatagcaa aagaggaaaa aacgtgcctg ttggattctt 300
cttcgtcgat agcctggcaa aataaaaaga aaccaaaatc aaaattgaaa aatcaataag 360
aaaaaataac tattattaag aagaaaacat gcataacaac aaataaaatt aaaaccttcc 420
aaatagaagc aaatatcaac gaaacaaaca tgcataacag taaaaaagga aaaaactttc 480
ccgttggatg ctttttcatt gatagcctgg gaaaattaaa aggaacctca tttttggggg 540
aaggctaacc tcttctctac agcctaccag gtccatataa ccatagcaaa ttcttacaac 600
agcaagagat atattacaaa caatattacc tttcttgttt ttggtaacca aaccaaattc 660
ttatagtagc aagcttaatt ttaaaaatga tatattttca agagaggaag caaaaagaag 720
aaagattcat cgcaagctca tatccatcaa ggcataacaa tctcaacata tgcaatgagc 780
ttaaacacat ttagaatgtt tccacataca tatagtcatt ttcttggtaa cacccgtgca 840
tatgttgata gtagttatga atttttaaga ttaaaaacta aaacttcatt taggcatttc 900
atcaaacaca acttacaatc catatgggga agatgacgaa aaaattagcc cgggaaaata 960
aaaaaggaac caaaaacaga gttgaaaaat cagtaagaaa aaataactat tattaagaag 1020
aaaacatgca taacaacaaa tcaaattaaa accttccaaa tagaagcaaa tatcaatgaa 1080
acaaacatgc ataacagtaa aaagggaaaa acctttgcga gaaacaggaa cgtttcggta 1140
ataggaactt gcgtgaatga tcctaacacc gattttcatg ttgggtcgag accaattgta 1200
agcgattgaa cgcgaaatac cactgagcca cagacaccct gatccaaata aaaatcgcaa 1260
aaaacacaaa aagatactaa gaggtgcaga gcttgcatgc aaatccagat aattaagaac 1320
tgaggaaaat gcataaaaac tcatagaatt tcctctaaaa gtatgaacaa aataaaaaat 1380
gagttttagg gaaaaacaac aacgtagcat aaaaaaacac aaaaaatcag acaaaaaacg 1440
atgatagaat aacaattaga gattggcgat aagcaaaagg ggcttacacg atcagtatgt 1500
atatcctttg taacctttga tggcttcacg aatctctttc ctgcagagga aaacgatgac 1560
aaattattga aagagataga aggaaggaaa aaaagagagg aaaaaagaca tgcacaacct 1620
cacattttgt attgcaaaaa gcgggtggct tcagcattct ggggaaattt cagtttccga 1680
aaaaaaaaaa gagaaaagag aaagagataa acagaggatg gtacgtggag ttggcagcaa 1740
aaaaaaaaag ttcataggaa aacacatggc agtgaggtgt cacaaaatca aagcaaagct 1800
gagggctata aaatgaccaa aaaataaaaa taaaaaatgg acaggtgtca agctctcaac 1860
tatgggtatt ttgaccaaaa aaccaaaatt ttggacagct gtcaaccttt cagtcatgga 1920
tatttcagta ttttccattt ccttctgatt tcatatatag atattgattt tcaaatatat 1980
tattcgtttt attttataca ttcttgaata aaaaaatggc accagacatg ttccactttc 2040
tgtttttaca tatatttaat atttaattta atatatataa tatttaatag aatatataat 2100
ttctcatttt ttaaggcatt attatttttc tgtggacccg aaatagatgc aggcgctggc 2160
tcgctcactc actaatcgcc ac 2182
<210> 12
<211> 22
<212> DNA
<213>Expand GmMDH12 cDNA overall lengths upstream specific primer (forward primer F)
<400> 12
ggatccatga tgaagccatc ga 22
<210> 13
<211> 27
<212> DNA
<213>Expand GmMDH12 cDNA overall lengths downstream special primer (reverse primer R)
<400> 13
acatgaattc ttactggttg gcaaatt 27
<210> 14
<211> 23
<212> DNA
<213>Expand GmMDH12 gene overexpression segments upstream specific primer (forward primer F)
<400> 14
acaaagagct cgagagagag aaa 23
<210> 15
<211> 28
<212> DNA
<213>Expand GmMDH12 gene overexpression segments downstream special primer (reverse primer R)
<400> 15
atatctagaa acctaccaga catgtatg 28

Claims (10)

1. a kind of soybeanGmMDH12Gene, which is characterized in that sequence is as shown in SEQ ID NO.3.
2. described in claim 1GmMDH12Gene coded protein, which is characterized in that amino acid sequence such as SEQ ID NO.4 institutes Show.
3. described in claim 1GmMDH12Application of the gene in terms of enhancing legume nodule malic acid synthesis.
4. described in claim 1GmMDH12Application of the gene in terms of promoting legume nodulation nitrogen fixing capacity.
5. described in claim 1GmMDH12The gene legume strong in cultivation root system Noduling ability and/or biological nitrogen fixation ability The application of aspect.
6. according to any application of claim 3~5, which is characterized in that the legume is soybean.
7. a kind of method of structure root system Noduling ability and/or the strong genetically engineered soybean of biological nitrogen fixation ability, which is characterized in that willGmMDH12Gene is overexpressed in soybean.
8. the method according to the description of claim 7 is characterized in that willGmMDH12Gene is connected on carrier PTF101S super It after measuring expression vector, is transferred in K599, and converted using the compound plant of agriculture bacillus mediated soybean transgene, obtains transgenosis Soybean.
9. according to the method described in claim 8, it is characterized in that, steps are as follows：
S1. overexpression carrier is built
Using soybean nodulation cDNA as template, with SEQ ID NO:14 and SEQ ID NO:Upstream and downstream specific primer PCR shown in 15 expands IncreaseGmMDH12Gene order；
After pcr amplified fragment recycling sequencing is errorless, pass throughSac IAnd XbaI is to amplified fragments and purpose carrier PTF101S difference After carrying out double digestion, amplified fragments are connected on purpose carrier PTF101S, expression vector is obtained；
S2. it converts：Then the expression vector built is transferred in K599 by freeze-thaw method, is turned using agriculture bacillus mediated soybean The compound plant of gene is converted, and the compound plant of transgenosis is obtained.
10. according to the method described in claim 9, it is characterized in that, the compound plant of obtained transgenosis need to pass through verification：Extraction The compound plant root of transgenosis and root nodule RNA, reverse transcription pass through quantitative PCR detection at cDNAGmMDH12Gene expression effect.
CN201810421922.8A 2018-05-04 2018-05-04 Application of GmMDH12 gene in promoting nodulation and nitrogen fixation of soybeans Active CN108795956B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810421922.8A CN108795956B (en) 2018-05-04 2018-05-04 Application of GmMDH12 gene in promoting nodulation and nitrogen fixation of soybeans

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810421922.8A CN108795956B (en) 2018-05-04 2018-05-04 Application of GmMDH12 gene in promoting nodulation and nitrogen fixation of soybeans

Publications (2)

Publication Number Publication Date
CN108795956A true CN108795956A (en) 2018-11-13
CN108795956B CN108795956B (en) 2021-04-23

Family

ID=64093329

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810421922.8A Active CN108795956B (en) 2018-05-04 2018-05-04 Application of GmMDH12 gene in promoting nodulation and nitrogen fixation of soybeans

Country Status (1)

Country Link
CN (1) CN108795956B (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109735561A (en) * 2019-03-18 2019-05-10 华中农业大学 A kind of breeding method of high nodulation and nitrogen fixation plant
CN114574518A (en) * 2022-02-09 2022-06-03 华中农业大学 Method for promoting nodulation of leguminous crops, especially salt-tolerant nodulation
CN116200404A (en) * 2023-04-10 2023-06-02 华南农业大学 Soybean asparagine synthetase analogous gene and application thereof

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101230357A (en) * 2007-12-05 2008-07-30 昆明理工大学 Plant expression vector for improving aluminum-tolerance of plant
CN101688196A (en) * 2007-03-20 2010-03-31 佛罗里达大学研究基金公司 Materials and methods for efficient succinate and malate production
CN102154322A (en) * 2011-03-14 2011-08-17 华南农业大学 Application of gene GmEXPB2 in improving phosphorus absorption efficiency of soybean plants
US20120260368A1 (en) * 2011-01-05 2012-10-11 Melissa Goellner Mitchum Genes Implicated In Resistance To Soybean Cyst Nematode Infection And Methods Of Their Use
CN104428414A (en) * 2012-05-02 2015-03-18 陶氏益农公司 Targeted modification of malate dehydrogenase
WO2016191293A1 (en) * 2015-05-22 2016-12-01 Board Of Regents, The University Of Texas System Prediction of hybrid vigor using circadian-regulated stress-responsive gene expression
WO2017190056A1 (en) * 2016-04-28 2017-11-02 William Marsh Rice University Conversion of 1-carbon compounds to products

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101688196A (en) * 2007-03-20 2010-03-31 佛罗里达大学研究基金公司 Materials and methods for efficient succinate and malate production
CN101230357A (en) * 2007-12-05 2008-07-30 昆明理工大学 Plant expression vector for improving aluminum-tolerance of plant
US20120260368A1 (en) * 2011-01-05 2012-10-11 Melissa Goellner Mitchum Genes Implicated In Resistance To Soybean Cyst Nematode Infection And Methods Of Their Use
CN102154322A (en) * 2011-03-14 2011-08-17 华南农业大学 Application of gene GmEXPB2 in improving phosphorus absorption efficiency of soybean plants
CN104428414A (en) * 2012-05-02 2015-03-18 陶氏益农公司 Targeted modification of malate dehydrogenase
WO2016191293A1 (en) * 2015-05-22 2016-12-01 Board Of Regents, The University Of Texas System Prediction of hybrid vigor using circadian-regulated stress-responsive gene expression
WO2017190056A1 (en) * 2016-04-28 2017-11-02 William Marsh Rice University Conversion of 1-carbon compounds to products

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHAE WOO LIM等: "Comparative proteomic analysis of soybean nodulation using a supernodulation mutant, SS2-2", 《BIOSCI.BIOTECHNOL.BIOCHEM》 *
IMSANDE J等: "Glycine max malate dehydrogenase (MDH-1),mRNA", 《GENBANK DATABASE》 *
SUSAN S. MILLER等: "Alfalfa malate dehydrogenase（MDH）：molecular cloning and characterization of five different forms reveals a unique nodu1e-enhanced MDH", 《THE PLANT JOURNAL》 *
汪清胤等: "大豆根瘤菌(Bradyrhizobium japonicum)苹果酸脱氢酶(mdh)基因的插入失活 ", 《大豆科学》 *
白振龙等: "苹果酸脱氢酶调控大豆苹果酸合成及根瘤生长的机制", 《中国优秀硕士学位论文全文数据库农业科技辑》 *
陈志坚等: "苹果酸脱氢酶(GmMDHl2)参与低磷调控大豆合成苹果酸的功能研究", 《2013全国植物生物学大会会议报告摘要》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109735561A (en) * 2019-03-18 2019-05-10 华中农业大学 A kind of breeding method of high nodulation and nitrogen fixation plant
CN114574518A (en) * 2022-02-09 2022-06-03 华中农业大学 Method for promoting nodulation of leguminous crops, especially salt-tolerant nodulation
CN114574518B (en) * 2022-02-09 2024-02-23 华中农业大学 Method for promoting nodulation, especially salt-tolerant nodulation, of leguminous crops
CN116200404A (en) * 2023-04-10 2023-06-02 华南农业大学 Soybean asparagine synthetase analogous gene and application thereof
CN116200404B (en) * 2023-04-10 2024-05-10 华南农业大学 Soybean asparagine synthetase analogous gene and application thereof

Also Published As

Publication number Publication date
CN108795956B (en) 2021-04-23

Similar Documents

Publication Publication Date Title
CN108728420A (en) It is a kind of regulation and control crop downgrade and its yield gene and its application
CN102757969A (en) Phosphorus transportprotein gene GmPT5 related to phosphorus transport of soybean nodulation and application thereof
CN108795956A (en) Application of the GmMDH12 genes in terms of promoting soybean nodulation nitrogen fixing capacity
CN104302773A (en) Methods of increasing tolerance to heat stress and amino acid content of plants
CN106591322A (en) Gingko MADS-box transcription factor gene GbMADS9 for controlling blossoming of plants and encoding protein and application of Gingko MADS-box transcription factor gene GbMADS9
CN113087804B (en) Bivalent plant immune fusion protein and production method and application thereof
CN112725349B (en) Dactylis glomerata zinc finger protein gene DgMYM1, and expression vector and application thereof
CN112322630B (en) MsSPL13 gene and application thereof
CN106978424A (en) A kind of drought-resistant maize related gene Zmhdz12 and its application
CN105524152B (en) Rice high-temperature resistant gene and application thereof in crop high-temperature resistant breeding
CN113621074B (en) Multivalent plant immune fusion protein and production method and application thereof
CN110229818A (en) Wax plum CpSNAC1 gene promoter and its application
JP4677568B2 (en) Production method of plants that grow nodules with high nitrogen fixation activity
KR102065491B1 (en) A root hair-specific promoter from Oryza sativa Os11g41640 gene and uses thereof
CN104745560A (en) Eggplant chalcone synthase SmCHS1 protein and coding gene thereof
CN101492689A (en) Corn C4 phosphoric acid enol pyruvic acid carboxylase gene and uses in producing wheat
CN111979253A (en) TrFQR1 gene, clone thereof, expression vector construction method and application
CN109354618A (en) Application of the G-protein α subunit in regulation germination of cucumber seeds, growth of seedling and plant low temperature resistivity
CN112708603B (en) Application of rice ARE2 gene in plant nitrogen metabolism regulation
CN104031927A (en) Gene OsPRO related to content of fragrance of fragrant rice and application of encoding protein of gene OsPRO
JP5531290B2 (en) gene
CN101679968B (en) Polypeptide capable of improving tolerance to iron deficiency in plant, and use thereof
CN109006917B (en) Application of wheat catalase in improving flour processing quality
Batcho et al. Transient expression analysis of Agave sisalana heat shock protein gene (AsHSP70) in model species (Nicotiana benthamiana) under heat stress
CN108276481B (en) Upland cotton GhLEA3 gene and application thereof in low-temperature stress resistance

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant